2-deoxy-3-phosphonylmethyl nucleosides

ABSTRACT

The invention relates to 2&#39;-deoxy-3&#39;-phosphonylmethyl nucleosides that are not substituted in the 2&#39;-position.

Phosphinylmethyl derivatives of nucleosides which carry a hydroxy groupin the 2'-position of the molecule are already known; see J. Am. Chem.Soc. 92 (1970) 5510.

The aim of the invention is to provide biologically active compoundswhich, inter alia, after customary modification and chemicalincorporation into an oligonucleotide sequence and further incorporationinto DNA, can no longer be cleaved by nucleases, especially byrestriction enzymes. To this end, according to the invention2'-deoxy-3'-phosphonylmethyl nucleosides of the following generalformula are provided: ##STR1## in which the following symbols have thefollowing meanings: B=adenine, N⁶ -benzoyladenine;

guanine, N² -isobutyryl guanine, N² -isobutyryl-4-O-(p-nitrophenylethyl)-guanine;

cytosine, N⁴ -benzoyl cytosine, 4-anisoyl cytosine;

thymine, 4-O-(p-nitrophenylethyl)-thymine; or uracil, and

(a)

X¹, X² =H, alkali metal, NH₄ or C₁₋₄ -alkyl; or

X¹ =chlorophenyl, 2,2,2-trichloroethyl or cyanoethyl and

X² -triethylammonium

R=OH, N₃, NH₂, NHR¹, NR¹ R², --O--PO(OY¹)(OY²),

--O--PS(OY¹)(OY²),

--O--PO(OY¹)--O--PO(OY²)(OY³),

--O--PO(OY¹)--O--PO(OY²)--O--PO(OY³)(OY⁴)

R¹, R² =C₁₋₄ -alkyl, C₄₋₇ -cycloalkyl, C₆₋₈ -alkylaryl or C₆₋₈-arylalkyl, and

Y¹, Y², Y³, Y⁴ =H, alkali metal or NH₄, or

R=5'-O-monomethoxytriphenylmethyl(5'-O-monomethoxytrityl) or5'-O-(4,4'-dimethoxytriphenylmethyl) (5'-O-(4,4'-dimethoxytrityl)) or

(b)

X¹ =H, alkali metal, NH₄ or C₁₋₄ -alkyl,

X² and R being absent with the formation of a ring of the grouping--PO(OX¹)--O--CH₂ --.

(I) Manufacture of compounds of the general formula according to claim 1in which the following symbols have the following meanings:

B=adenine, R=OH, X¹ and X² =H, alkali metal or NH₄ ; or

B=adenine, R=OH, X¹ and X² =C₁₋₄ -alkyl; or

B=adenine, R=OH, X¹ =H, alkali metal or NH₄ and X² =C₁₋₄ -alkyl.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing of an embodiment synthesis of theinvention as set forth in the examples.

FIG. 2 is a representation of a thin-layer chromatogram and itsinterpretation, in reference to the examples.

DETAILED DESCRIPTION OF THE INVENTION

The compounds according to the invention can be manufactured accordingto the invention by

(a) using a compound of the general formula 1 ##STR2## as startingmaterial (Z=benzoyl or monomethoxytrityl), which has been obtained inaccordance with Moffatt in the modification of Vorbruggen [see J. Am.Chem. Soc. 92 (1970) 5511 and Chem. Ber. 114 (1981) 1279]. From thiscompound of the general formula 1 it is possible to remove selectivelythe acetyl group and the benzoyl group in the 6-position using aninorganic N-base or one of the salts thereof (for example withhydroxyammonium acetate in pyridine) in accordance with Ishido et al.[J. C. S. Perkin 1 (1979) 2088 or 1 (1980) 563].

(b) Subsequently the reaction product of the general formula 2 ##STR3##can be esterified with a compound of the formula R³ --CS--R⁴ (R³ =Cl, R⁴=phenyl, phenoxy; R³ =R⁴ =imidazol-1-yl) in the presence of4-(dialkylamino)-pyridine (for example 4-(dimethylamino)-pyridine) in anorganic solvent (for example dichloromethane) [see J. Org. Chem. 46(1981) 4300 and 4843 and J. Am. Chem. Soc. 103 (1981) 932 andreferences].

(c) The resulting reaction product of the general formula 3 ##STR4## inwhich A represents an adenine radical, can be reduced with tributylinhydride in toluene, preferably at elevated temperature, especially inthe range of from 30° to 90° C., for example at approximately 60° C.

(d1) The reaction product of the general formula 4 ##STR5## can betransesterified with halotrimethylsilane (such as bromo- oriodo-trimethylsilane) in a halogenated hydrocarbon (such asdichloromethane) and hydrolysed to form the free acid or its ammonium oralkali metal salt of the general formula of claim 1 (in which X¹ and X²represent a hydrogen atom, an alkali metal or NH₄ and R represents an OHgroup), or

(d2) the mentioned protecting group Z (Z=benzoyl) can be removed fromthe reaction product of the general formula 4 with esterase(E.C.3.1.1.1) from pig's liver to form a diester of the general formulaaccording to claim 1 (X¹ and X² representing a C₁₋₄ -alkyl group and Rrepresenting an OH group), and, optionally,

(d3) the diester can be hydrolysed to form a monoester of the generalformula according to claim 1 (X¹ representing a hydrogen atom, an alkalimetal or NH₄, X² representing a C₁₋₄ -alkyl group and R representing anOH group).

Stage (a) can be carried out with a yield of from 60 to 70%, and stages(b), (c), (d1), (d2) and (d3) can be carried out with virtuallyquantitative yields. (II) Manufacture of compounds of the generalformula according to claim 1 in which the following symbols have thefollowing meanings:

B=adenine, R=N₃ and X¹ and X² =C₁₋₄ -alkyl; or

B=adenine, R=NH₂, NHR¹ or NHR² (R¹, R² =C₁₋₄ -aliphatic, C₄₋₇-cycloaliphatic, C₆₋₈ -aliphatic/aromatic or C₆₋₈ -aromatic/aliphaticradical), X¹ =H, alkali metal or NH₄ and X² =C₁₋₄ -alkyl; or

B=adenine, R=N₃, NH₂, NHR¹ or NR¹ R² (R¹, R² =C₁₋₄ -aliphatic, C₄₋₇-cycloaliphatic, C₆₋₈ -aliphatic/aromatic or C₆₋₈ -aromatic/aliphaticradical) and X¹ and X² =H, alkali metal or NH₄.

The compounds according to the invention can be manufactured accordingto the invention as follows:

(a) there is used as starting material a diester according to (I)(d2)which is activated in the 5'-position, for example with tosyl chloride(p-toluenesulphonic acid chloride) in pyridine.

(b1) Thereafter, the activated diester can be reacted with an azide, forexample sodium azide, in dimethylformamide, to form a diester of thegeneral formula according to claim 1 (B representing an adenosineradical, R representing an azide radical and R¹ and R² representing aC₁₋₄ -alkyl radical); or

(b2) the activated diester can be reacted with liquid ammonia or with aprimary or secondary amine to form a semiester of the general formulaaccording to claim 1 (B representing an adenosine radical, Rrepresenting an NH₂, NHR¹ or NR¹ R² radical, R¹ and R² representing aC₁₋₄ -aliphatic, C₄₋₇ -cycloaliphatic, C₆₋₈ -aliphatic/aromatic or C₆₋₈-aromatic/aliphatic radical, X¹ representing a hydrogen atom, an alkalimetal or NH₄, and X² representing a C₁₋₄ -alkyl radical); and,optionally,

(b3) the resulting diester or semiester can be transesterified with ahalotrimethylsilane (for example bromo- or iodo-trimethylsilane) andhydrolysed to form the free acid or one of the salts thereof of thegeneral formula according to claim 1 (B representing an adenosineradical, R representing an N₃, NH₂, NHR¹ or NR¹ R² radical, R¹ and R²representing a C₁₋₄ -aliphatic, C₄₋₇ -cycloaliphatic, C₆₋₈-aliphatic/aromatic or C₆₋₈ -aromatic/aliphatic radical and X¹ and X²representing a hydrogen atom, an alkali metal or NH₄).

(III) Manufacture of compounds of the general formula according to claim1 in which the following symbols have the following meanings:

B=adenine, R=--O--PO(OY¹)(OY²), --O--PS(OY¹)(OY²),--O--PO(OY¹)--O--PO(OY²)(OY³) or--O--PO(OY¹)--O--PO(OY²)--O--PO(OY³)(OY⁴) (Y¹, Y², Y³ and Y⁴ =H, alkalimetal or NH₄) and X¹ and X² =C₁₋₄ -alkyl; or

B=adenine, R=--O--PO(OY¹)(OY²), --O--PS(OY¹)(OY²),--O--PO(OY¹)--O--PO(OY²)(OY³) or--O--PO(OY¹)--O--PO(OY²)--O--PO(OY³)(OY⁴) (Y¹, Y², Y³ and Y⁴ =H, alkalimetal or NH₄), X¹ =H, alkali metal or NH₄ and X² =X¹ or C₁₋₄ -alkyl.

The compounds according to the invention can be manufactured accordingto the invention as follows:

(a) there is used as starting material a diester according to (I)(d2)which is phosphorylated according to Yoshikawa et al. [Tetrahedron Lett.(1967) 5065], for example with POCl₃ or PSCl₃ in a phosphoric acidtrialkyl ester (such as triethyl phosphate) to form a diester of thegeneral formula according to claim 1, and hydrolysed to form the free5'-monophosphoric or 5'-thionophosphoric acid or salts thereof (Brepresenting an adenosineradical, R representing a --O--PO(OY¹)(OY²) or--O--PS(OY¹)(OY²) radical, Y¹ and Y² representing a hydrogen atom, analkali metal or NH₄ and X¹ and X² representing a C₁₋₄ -alkyl radical);and, optionally,

(b) the resulting product is hydrolysed to form a semiester or to formfree 3'-methylenephosphonic acid of the general formula according toclaim 1 (B representing an adenosine radical, R representing a--O--PO(OY¹)(OY²) or --O--PS(OY¹)(OY²) radical, X¹, Y¹ and Y²representing a hydrogen atom, an alkali metal or NH₄ and X² =X¹ or aC₁₋₄ -alkyl radical); and, optionally.

(c) before or after step (b), the phosphoric acid obtained in stage (a)or its salt is phosphorylated according to Hoard & Ott [J. Am. Chem.Soc. 87 (1965) 1785] or Michelson [Biochim. Biophys. Acta, 1 (1964) 91]or enzymatically according to Marutsky (Diss. TU Braunschweig 1975), toform di- or tri-phosphoric acids or salts thereof of the general formulaaccording to claim 1 (B representing an adenosine radical, Rrepresenting a --O--PO(OY¹)--O--PO(OY²)(OY³) or--O--PO(OY¹)--O--PO(OY²)--O--PO(OY³)(OY⁴) radical, Y¹, Y², Y³ and Y⁴representing a hydrogen atom, an alkali metal or NH₄ and X¹ representinga hydrogen atom, an alkali metal, NH₄ or a C₁₋₄ -alkyl radical, and X²representing a C₁₋₄ -alkyl radical).

(IV) Manufacture of compounds of the general formula according to claim1 in which the following symbols have the following meanings:

B=adenine, R and X² are absent with the formation of a ring of thegrouping --PO(OX¹)--O--CH₂ --, X¹ =H, alkali metal, NH₄ or C₁₋₄ -alkyl.

The compounds according to the invention can be manufactured accordingto the invention as follows:

(a) there is used as starting material a diester according to (I)(d2)which is reacted with alkaline catalysts to form a compound of thegeneral formula according to claim 1 (B representing an adenosineradical and X¹ representing a C₁₋₄ -alkyl radical and R and X² beingabsent with the formation of a ring of the grouping --PO(OX¹)--O--CH₂--); or

(b) the free acid or one of its salts according to (I)(d1) is used asstarting material and this starting compound (optionally in the form ofits pyridinium salt) is cyclised with cyclisation agents (for exampledicyclohexyl carbodiimide) in an organic solvent (for example pyridine)at elevated temperature; and, optionally,

(c) the resulting product of stage (a) is transesterified with ahalotrimethylsilane (for example bromo- or iodo-trimethylsilane) in ahalogenated hydrocarbon (for example dichloromethane) and hydrolysed toform the free acid or salts thereof of the general formula according toclaim 1 (B representing an adenosine radical and X¹ representing ahydrogen atom, an alkali metal or NH₄ and R and X² being absent with theformation a ring of the grouping --PO(OX¹)--O--CH₂ --.

Compounds of the general formula according to claim 1 in whichB=guanine, cytosine, thymine or uracil can be manufactured by using asstarting materials, instead of compounds of the general formula 2,corresponding compounds that carry a guanine, cytosine, thymine oruracil radical in the 1'-position.

(V) Manufacture of compounds of the general formula according to claim 1in which the following symbols have the following meanings:

R=5'-O-monomethoxytrityl or 5'-O-(4,4'-dimethoxytrityl)

B=N⁶ -benzoyladenine; N² -isobutyryl guanine, N²-isobutyryl-4-O-(p-nitrophenylethyl)-guanine;4-O-(p-nitrophenylethyl)-thymine; N⁴ -benzoyl cytosine or 4-anisoylcytosine

X¹ =2-chlorophenyl, 2,2,2-trichloroethyl or cyanoethyl

X² =triethylammonium

These inventive compounds, for example compounds having the generalformula 7 ##STR6## can be prepared as follows:

(a) A compound having the general formula 8 and prepared as describedbefore under (I) where the protecting group Z has been cleaved in step(d2) ##STR7## can be reacted with4-monomethoxytriphenylmethylchloride(methoxytritylchloride) in thepresence of 4-dimethylaminopyridine; cf. Y Lapidot and H. G. Khorana, J.Am. Chem. Soc. 85, 3862 (1963).

(b) The resulting compound having the general formula 9 ##STR8## can bereacted with a halogenotrimethylsilane, viz.

(b1) with chlorotrimethylsilane in the presence of sodium iodide andacetonitrile according to T. Morita, Y. Okamoto and H. Sakurai,Tetrahedron Letters, No. 28, pages 2523 to 2526 (1978) or

(b2) with bromotrimethylsilane according to C. E. McKenna, H. T. Higa,N. H. Higa and M. McKenna, Tetrahedron Letters, 977, (1977).

(c) The reaction product having the general formula 10 ##STR9## ishydrolized with methanol and subjected to a chromatography onDEAE-sephadex.

(d) The reaction product having the general formula 11 ##STR10## is thenesterified in a manner known per se with 2-chlorophonol using2,4,6-triisopropylbenzenesulfochloride in pyridine (activation) toobtain a compound having the general formula 7.

The inventive compounds can be used in connection with a really chemicalsynthesis of oligonucleotides according to the phosphotriester method:

The synthesis method was tested with isobutylphosphonic acid monoesterof deoxyadenosine as model compound: ##STR11## (alpha)isobutylphosphonic acid having the formula 13 ##STR12## is reacted in amanner known per se with triisopropylbenzenesulfochloride in pyridinewith 2-chlorophenol and then subjected to a chromatography onDEAE-sephadex.

(beta) The reaction product having the formula 14 ##STR13## is reactedwith MSNT (1-(mesitylenesulfonyl)-3-nitro-1,2,4-triazole) ascondensation agent in pyridine and deoxy-3'-benzoyloxy-N⁶-benzoyladenosine.

(gamma) The resulting mixed isobutylphosphonic acid diester having theformula 15 ##STR14## is hydrolized with concentrated ammonia todeoxyadenosineisobutylphosphonic acid monoester having the formula 12.

The invention will be described in more detail in the following by wayof examples with reference to the accompanying drawings of FIGS. 1 and 2showing the structure of the compounds 1-6, referred to.

9-[5-O-Benzoyl-3-deoxy-3-(diethoxyphosphonylmethyl)-β-D-ribofuranosyl]-adenineor (5'-O-Benzoyl-3'-diethoxyphosphonylmethyl-3'-deoxyadenosine) (2)

1.134 g (1.74 mmol) of 1 are dissolved in 35 ml of pyridine withapproximately 9 mmol of hydroxyammonium acetate (NH₂ OH.HAc) and areleft to stand at room temperature for 45 hours.

Examination of a sample by thin-layer chromatography and subsequentevalution using a TLC-scanner gives the result shown in FIG. 2 of theaccompanying drawings.

After removal of the volatile components (pyridine and excesshydroxyammonium acetate) under an oil pump vacuum, the residue isdissolved in chloroform/methanol 9:1 and introduced onto a column ofsilica gel (25×5 cm, silica gel 60, particle size 0.063-0.200 mm, Merck,Darmstadt). After elution with the same eluant and concentration byevaporation of the peak containing 2 there is obtained 580 mg(approximately 65%). Elution with methanol yields the semiester of 2.

9-[5-O-Benzoyl-3-deoxy-3-(diethoxyphosphonylmethyl)-2-O-phenoxythiocarbonyl-β-D-ribofuranosyl]-adenine(3) or5'-O-Benzoyl-3'-diethoxyphosphonylmethyl-2'-O-phenoxy-thiocarbonyl-3'-deoxyadenosine

420 mg (0.83 mmol) of 2 are dissolved in 20 ml of anhydrousdichloromethane (Robins et al., operation in acetonitrile is notpossible in the above case because the starting material crystallisesout after a prolonged period) and, with the exclusion of moisture, 0.21ml (1.5 mmol) of thiocarbonic acid O-phenyl ester chloride and 270 mg(2.2 mmol) of 4-dimethylaminopyridine are added. After stirring at roomtemperature for 12 hours, the reaction is complete (TLC, silica gel,eluant: dichloromethane/methanol 9:1). The mixture is concentrated todryness and the residue is partitioned between ethyl acetate (EA) and 1MKH₂ PO₄ solution (pH 4). After drying the EA phase with anhydrous sodiumsulphate, it is concentrated to dryness and the residue is dissolved ina small quantity of dichloromethane/methanol 9:1 and chromatographedover a column of silica gel (10×5 cm). After concentration of thefraction containing 3 there is obtained 445 mg (67%).

9-[2-Deoxy-5-O-benzoyl-3-deoxy-3-(diethoxyphosphonyl-methyl)-β-D-ribofuranosyl]-adenine(4) or5'-O-Benzoyl-3'-deoxy-3'-diethoxyphosphonylmethyl-2'-deoxyadenosine

190 mg (0.29 mmol) of 3 are dissolved in 6 ml of anhydrous toluene, and0.155 ml of tributyltin hydride and 32 mg of2,2'-azo-bis(2-methylpropionitrile) or (α,α-azo-isobutyronitrile) areadded. After heating at 70° C. with the exclusion of moisture, thereaction is complete after 7 hours. After concentration to dryness theresidue is dissolved in a small quantity of chloroform/methanol 9:1 andthe solution is introduced onto a column of silica gel (24×2 cm). Afterelution with the above eluant, the peak containing 4 is concentrated.Yield: 123 mg (86%).

9-[2-Deoxy-3-deoxy-3-(dihydroxyphosphonylmethyl)-β-D-ribofuranosyl]-adenineor 2'-Deoxy-3'-dihydroxyphosphonylmethyl-3'-deoxyadenosine (5)

166 mg (0.34 mmol) of 4 are dissolved in 4 ml of anhydrousdichloromethane, and 0.27 ml (1.7 mmol) of trimethylbromosilane areadded. The mixture is stirred overnight (in TLC, eluant:chloroform/methanol 9:1, starting material was no longer present) andconcentrated in vacuo. The residue is hydrolysed with water, and theaqueous weakly acidic solution is concentrated to dryness. In order toremove the 5'-O-benzoyl protecting group, the residue is left to standin saturated methanolic ammonia solution (25 ml) for 5 days. Afterevaporation to dryness the residue is dissolved in 10 ml of water,adjusted to pH 7.5, introduced onto a DEAE Sephadex column (HCO₃ -form,30×25 cm) and chromatographed with a gradient of 1 liter of water/1liter of 0.4M TEAB buffer. After concentration of the peak containing 5and repeated concentration by evaporation with methanol, the aqueoussolution is introduced onto a Dowex sodium columnn and then lyophilised.

9-[2-Deoxy-3-deoxy-3-(diethoxyphosphonylmethyl)-β-D-ribofuranosyl]-adenineor 2'-Deoxy-3'-diethoxyphosphonylmethyl-3'-deoxyadenosine (6)

8 mg (16 μmol) of 4 are dissolved in 4 ml of water, and the solution isadjusted to pH 8.2 with saturated sodium bicarbonate solution. After theaddition of 10 μl of esterase (Boehringer) the mixture is stirred for 24hours at 37° C. while monitoring the pH value. The reaction is followedusing thin layer chromatography (TLC, silica gel, eluant:chloroform/methanol 8:2). After concentration to dryness and extractionusing the eluant, chromatography is carried out over a small column ofsilica gel. Yield: 5.2 mg (83%).

The structures of all compounds are ascertained accurately by ¹ H, ¹³ Cand ³¹ P spectra and by chromatographic tests.

Abbreviations:

TLC=thin layer chromatography

TEAB buffer=triethylammonium bicarbonate buffer

We claim:
 1. A 2'-deoxy-3'-phosphonylmethyl nucleoside of the formula##STR15## in which B is attached to the C-1 carbon of the deoxyribose inthe β configuration and is selected from the group consisting ofadenine, N⁶ -benzoyladenine, guanine, N² -isobutyryl guanine, N²-isobutyryl-4-O-(p-nitrophenylethyl)-guanine, cytosine, N⁴ -benzoylcytosine, 4-anisoyl cytosine, thymine, 4-O-(p-nitrophenylethyl)-thymine,and uracil,X¹ and X² are selected from the group consisting of H, alkalimetals, NH₄ and C₁₋₄ -alkyl; or X² is triethylammonium and X¹ isselected from the group consisting of chlorophenyl, 2,2,2-trichloroethyland cyanoethyl, and R is selected from the group consisting of OH,benzoyl, N₃, NH₂, NHR¹, NR¹ R², --O--PO(OY¹)(OY²), --O--PS(OY¹)(OY²),--O--PO(OY¹)--O--PO(OY²)(OY³), and--O--PO(OY¹)--O--PO(OY²)--O--PO(OY³)(OY⁴) in whichR¹ and R² are selectedfrom the group consisting of C₁₋₄ -alkyl, C₄₋₇ -cycloalkyl, C₆₋₈-alkylaryl and C₆₋₈ -aryalkyl, and Y¹ Y², Y³ and Y⁴ are selected fromthe group consisting of H, alkali metals and NH₄, or R is selected fromthe group consisting of 5'-O-monomethoxytriphenylmethyl and5'-O-(4,4'-dimethoxytriphenylmethyl).
 2. A nucleoside according to claim1, in which B is selected from the group consisting of adenine, N⁶-benzoyladenine, guanine, N² -isobutyryl guanine, cytosine, N⁴ -benzoylcytosine, 4-anisoyl cytosine, thymine and uracil.
 3. A nucleosideaccording to claim 1, in which X¹ and X² are selected from the groupconsisting of H and C₁₋₄ -alkyl.
 4. A nucleoside according to claim 1,in which X² is triethylammonium and X¹ is selected from the groupconsisting of chlorophenyl, 2,2,2-trichloroethyl and cyanoethyl.
 5. Anucleoside according to claim 4, in which R is selected from the groupconsisting of 5'-O-monomethoxytriphenylmethyl and5'-O-(4,4'-dimethoxytriphenylmethyl.
 6. A nucleoside according to claim4, in which R is selected from the group consisting of benzoyl,--O--PO(OY¹)(OY²), --O--PO(OY¹)--O--PO(OY²)(OY³), and--O--PO(OY¹)--O--PO(OY²)--O--PO(OY³)(OY⁴) in whichY¹, Y², Y³ and Y⁴ areselected from the group consisting of H, alkali metals and NH₄.
 7. Anucleoside according to claim 1, in which B is selected from the groupconsisting of adenine, guanine, cytosine and thymine.
 8. A nucleosideaccording to claim 7, in which X¹ and X² are selected from the groupconsisting of H, alkali metals and NH₄.